Multi-omics and experimental validation identify methylation-related genes and METTL16 as key regulators in diabetic foot ulcer pathogenesis

Diabetic foot ulcers (DFUs) are a severe complication of diabetes, characterized by impaired wound healing, chronic inflammation, and tissue degradation. N⁶-methyladenosine (m⁶A), has emerged as a critical regulator in gene expression and cellular function in wound healing. This study aimed to systematically investigate the role of methylation-related genes (MRGs) in DFU pathogenesis, focusing on their diagnostic and therapeutic potential through integrative multi-omics analysis and experimental validation. Publicly available bulk RNA-seq, microarray, and single-cell RNA sequencing (scRNA-seq) datasets were analyzed to identify differentially expressed MRGs (DE-MRGs). Machine learning algorithms (LASSO, GBM, SVM-RFE, Random Forest) were used to screen key biomarkers. Immune infiltration and pathway enrichment analyses characterized inflammatory signatures, while scRNA-seq mapped MRG dynamics across cell types. Functional assays validated the role of METTL16 in high glucose-treated human skin fibroblasts (HSFs), assessing collagen synthesis, oxidative stress, and cellular migration. Thirteen DE-MRGs were identified in DFU tissues, with METTL16, NSUN3, and IGF2BP2 prioritized as diagnostic biomarkers. Immune profiling revealed M1 macrophage enrichment and dysregulated IL-17/MAPK pathways. Single-cell analysis highlighted METTL16's dynamic role in fibroblast activation and intercellular communication. Cell-based experiments confirmed the protective effects of METTL16 in HSFs, including enhanced migration, restored collagen synthesis, and reduced oxidative stress under hyperglycemic conditions. This study systematically identifies MRGs as critical regulators in DFU pathogenesis, with METTL16 emerging as a promising diagnostic biomarker and therapeutic target. The findings provide novel insights into the molecular mechanisms underlying DFU progression and offer potential avenues for improved diagnostic tools and therapeutic strategies.